1. Field of the Invention
The present invention relates to a reagent for analyzing leucocytes, especially for classifying and counting leucocytes using a classifying apparatus for leucocytes in the field of a clinical test.
2. Description of the Prior Art
In the field of a clinical test, it is important for the diagnosis of various diseases to classify and count leucocytes using the whole blood of patients.
For such a purpose, many apparati and methods have been reported.
In those apparati, leucocytes are classified into their subgroups (lymphocytes, monocytes, neutrophils, eosinophils and basophils) by the difference of RF signal intensity (changes in electric impedances at high-frequency), DC signal intensity (changes in direct current caused by the difference in the electroconductivity between the suspended particles and the liquid medium in which the particles are suspended), intensity of fluorescence, intensity of scattered light, absorbance, depolarization of scattered light or the like. In the classification and counting of leucocytes, it is necessary to pretreat the blood and lyse the erythrocytes so that the difference among each subgroup of the leucocytes according to the above-mentioned parameters can be detected.
For such a purpose, for example, leucocytes are shrunk to a suitable size using a lysing agent or specific cells are stained. The lysing agent used in shrinking leucocytes is an organic carboxylic acid, organic sulfonic acid or phenol (WO88/07187), or two kinds of cationic surfactants (WO 84/03771 and WO 84/02777). Examples of methods for lysing erythrocytes and staining specific cells are a method in which the blood is treated with a reagent comprising the combination of an erythrocyte lysing agent (saponin and/or sodium dodecylsulfate), a tertiary or quaternary ammonium salt, an alcohol, a polyoxyethylene sorbitan ester, glutaraldehyde or formaldehyde, alkylene glycol, a physiological salt and Chlorazol Black (Japanese Laid-Open Patent Publication Hei-03/266999 (corresponding to: FR8915166)) and a method in which blood is treated with a reagent comprising the combination of sodium dodecylsulfate, formaldehyde, sugar (or sugar alcohol) and a buffer, followed by subjecting to a peroxidase staining (Japanese Laid-Open Patent Publication Sho-62/071857 (corresponding to: U.S. Pat. Nos. 4,801,549 and 4,978,624)).
In the above-mentioned WO 84/03771 and WO 84/02777, the classification and counting of leucocytes are carried out by means of the difference in the DC signal intensity, which is convenient but has a disadvantage that leucocytes can be only classified into three subgroups.
Methods according to Japanese Laid-Open Patent Publication Sho-62/071857 and Hei-03/266999 can classify leucocytes into four or more subgroups, but require a treatment at a high temperature and a fixation using a toxic aldehyde.
Further, in the method of WO88/07187, leucocytes are detected by means of a RF signal, DC signal or the intensity of scattered light which has, however, a drawback in that the detectors are complicated, large in size and expensive.